Enzyme Fraction Research Articles

Effects of soil properties and management on the activity of soil organic matter transforming enzymes and the quantification of soil-bound and free activity

The decomposition of soil organic matter is mediated by extracellular enzymes. The aim of this work was to identify the factors determining the activity and size of the mobile fraction of extracellular enzymes (laccase, Mn-peroxidase, endocellulase, cellobiohydrolase, β-glucosidase, endoxylanase, β-xylosidase, α-glucosidase, chitinase, arylsulfatase, phosphatase, phosphodiesterase, alanine and leucine aminopeptidase) using a set of soils covering a wide range of physico-chemical properties. Organic matter content had a major effect on enzyme activity both in forest and grassland soils, while the effects of pH and humic compounds content were only important in forest soils, and the molecular mass of humic compounds and Ca content were only important in grasslands. Specific enzyme activity was either comparable between the soil types or higher in grasslands. With the exception of Mn-peroxidase and β-glucosidase, the specific activities of all enzymes in arable fields under tillage were similar to those in grasslands. Mobility differed among the enzymes and ranged from <1% for arylsulfatase and phosphodiesterase up to 20–40% for α-glucosidase and aminopeptidases, with pH being the most important variable. These results demonstrate that the factors regulating enzyme activity are likely to be different in forest soils and grasslands and that enzyme mobility is a characteristic feature of each individual enzyme.

Point Mutations (Q19P and N23K) Increase the Operational Solubility of a 2α-O-Benzoyltransferase that Conveys Various Acyl Groups from CoA to a Taxane Acceptor

Two site-directed mutations within the wild-type 2-O-benzoyltransferase (tbt) cDNA, from Taxus cuspidata plants, yielded an encoded protein containing replacement amino acids at Q19P and N23K that map to a solvent-exposed loop region. The likely significant changes in the biophysical properties invoked by these mutations caused the overexpressed, modified TBT (mTBT) to partition into the soluble enzyme fraction about 5-fold greater than the wild-type enzyme. Sufficient protein could now be acquired to examine the scope of the substrate specificity of mTBT by incubation with 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III that was mixed individually with various substituted benzoyls, alkanoyls, and (E)-butenoyl CoA donors. The mTBT catalyzed the conversion of each 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III to several 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues. The relative catalytic efficiency of mTBT with the 7,13-O,O-diacetyl-2-O-debenzoyl surrogate substrate and heterole carbonyl CoA substrates was slightly greater than with the natural aroyl substrate benzoyl CoA, while substituted benzoyl CoA thioesters were less productive. Short-chain hydrocarbon carbonyl and cyclohexanoyl CoA thioesters were also productive, where C(4) substrates were transferred by mTBT with approximately 10- to 17-fold greater catalytic efficiency compared to the transfer of benzoyl. The described broad specificity of mTBT suggests that a plethora of 2-O-acyl variants of the antimitotic paclitaxel can be assembled through biocatalytic sequences.

Application of XANES spectroscopy in understanding the metabolism of selenium in isolated rainbow trout hepatocytes: insights into selenium toxicity

Selenium (Se) is an essential element, but causes toxic effects in fish at a slightly elevated level beyond the threshold. However, the degree of Se toxicity differs depending on the chemical forms of Se (e.g., organic vs. inorganic) to which fish are exposed to. The mechanisms of Se metabolism and toxicity in fish, particularly at cellular level, are poorly understood. The present study was designed to examine the metabolic fate of different seleno-compounds, both inorganic and organic, in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) in primary culture using XANES spectroscopy. In cells exposed to 100 μM of selenate and selenite for 6-24 h, elemental Se was found to be the primary metabolite. Whereas, selenocystine appeared to be the major metabolite in cells exposed to 100 μM seleno-L-methionine for 6-24 h. Interestingly, we recorded L-methionine-γ-lyase activity in S9 fraction of cell lysate-an enzyme that directly catalyzes selenomethionine into methylselenol. We also found concurrent reduction of glutathione (GSH) concentration following reaction of seleno-L-methionine with cellular S9 fraction. Moreover, we observed a rapid increase in cellular reactive oxygen species (ROS) generation with increasing seleno-L-methionine exposure dose (100-1000 μM). These findings indicated the rapid cellular metabolism of seleno-L-methionine into methylselenol at higher exposure dose (≥100 μM), and the occurrence of GSH mediated redox cycling of methylselenol--a process that is known to produce reactive oxygen species (ROS). Overall, our results suggest that inorganic and organic selenium are metabolized through different metabolic pathways in rainbow trout hepatocytes. The findings of our study have important implications for understanding the chemical species-specific differences in Se toxicity to fish.

Binding Kinetics and Fraction of Immobile Enzymes Bound to Cellulose Fibrils Studied Through Confocal Laser Scanning Fluorescence Microscopy and FRAP

Biofuels and bioproducts derived from cellulosic biomass represent great potential renewable and environmentally friendly technologies. Key to converting cellulosic biomass into soluble sugars is the depolymerization of the cellulose macromolecules by enzymes called cellulases. These enzymes depolymerize the cellulose chain by binding to the exposed cellulose surface and cleaving β-glucosidic bonds. Although much work has been done studying the dynamics of these enzymes in bulk solution, little is known about how these enzymes operate at the micron to nanoscale.

Optimisation of the Mashing Procedure for 100% Malted Proso Millet (Panicum miliaceum L.) as a Raw Material for Gluten-free Beverages and Beers

Proso millet is a gluten-free cereal and is therefore considered a suitable raw material for the manufacturing of foods and beverages for people suffering from celiac disease. The objective of this study was to develop an optimal mashing procedure for 100% proso millet malt with a specific emphasis on high amylolytic activity. Therefore, the influence of temperature and pH on the amylolytic enzyme activity during mashing was investigated. Size exclusion chromatography was used to extract different amylolytic enzyme fractions from proso millet malt. These enzymes were added into a pH-adjusted, cold water extract of proso millet malt and an isothermal mashing procedure was applied. The temperatures and pH optima for amylolytic enzyme activities were determined. The α-amylase enzyme showed highest activity at a temperature of 60°C and at pH 5.0, whereas the β-amylase activity was optimum at 40°C and pH 5.3. The limit dextrinase enzyme reached maximum activity at 50°C and pH 5.3. In the subsequent mashing regimen, the mash was separated and 40% was held for 10 min at 68°C to achieve gelatinisation. The next step in the mashing procedure was the mixture of the part mashes. The combined mash was then subjected to an infusion mashing regimen, taking the temperature optima of the various amylolytic enzymes into account. It was possible to obtain full saccharification of the wort with this mashing regimen. The analytical data obtained with the optimised proso millet mash were comparable to barley wort, which served as a control.

Human Milk Fat Substitute from Butterfat: Production by Enzymatic Interesterification and Evaluation of Oxidative Stability

AbstractRecent data have suggested that the fatty acid composition and molecular structure of fats in infant formulas should be as similar to human milk fat as possible to obtain optimal fat and calcium absorption from the infant formula. This work investigated the possibilities of using enzyme technology and butterfat as a material to produce a fat similar to human milk fat with respect to the above parameters. Moreover, the oxidative stability of the enzyme modified human milk fat substitute (HMFS) was compared to the fat blend used for the production of HMFS. Using a combination of enzyme technology, fractionation and batch deodorization and with butterfat in combination with soybean oil and rapeseed oil as raw materials it was possible to produce HMFS with a molecular structure and fatty acid composition that was very similar to that of human milk fat. The oxidative stability of the HMFS oil was lower than that of the reference oil with the same fatty acid composition. However, oxidation did not lead to a severe increase in rancidity scores during storage. Rather, the panel gave high intensity scores for other off‐flavors such as burnt and bitter. Further optimization of the deodorization process is therefore necessary to remove these off‐flavors.

PXII-6 Degradation an aggresome formation of the Gn tail of the apathogenic tula hantavirus

Aggresome formation in cells involves the failure of the ubiquitin-proteasome pathway to dispose of proteins destined for degradation by the 26S proteasome. UBB+1 is present in Mallory bodies in alcoholic liver disease and in aggresomes formed in Alzheimer’s desease. The present investigation focuses on the role that UBB+1 plays in cytokeratin aggresome formation in Mallory bodies (MBs) in vitro. Immunoprecipitation with a monoclonal antibody to cytokeratin-8 (CK-8) was used. The immunoprecipitate was incubated for 24 h in the presence of different constituents involved in aggresome formation including ubiquitin, UBB+1, the proteasome inhibitor PS341, an ATP generating energy source, a deubiquitinating enzyme inhibitor, a purified proteasome fraction, and an E1–3 conjugating enzyme fraction. MB-like protein aggregates formed in the presence of ubiquitin, plus UBB+1 or PS341. These aggregates stained positively for CK-8. UBB+1, and a proteasome subunit Tbp7, as demonstrated on Western blots. A second approach was used to form MBs in vitro in cultured hepatocytes transfected with UBB+1 protein using Chariot. The cells were double stained using CK-8 and ubiquitin antibodies. The two proteins colocalized in MB-like aggregates. The results support the possibility that aggresome formation is a complex multifactor process, which is favored by inhibition of the proteasome and by the presence of UBB+1.

PXII-4 Human papillomavirus type 16: a study on its variants in the L1 gene

Aggresome formation in cells involves the failure of the ubiquitin-proteasome pathway to dispose of proteins destined for degradation by the 26S proteasome. UBB+1 is present in Mallory bodies in alcoholic liver disease and in aggresomes formed in Alzheimer’s desease. The present investigation focuses on the role that UBB+1 plays in cytokeratin aggresome formation in Mallory bodies (MBs) in vitro. Immunoprecipitation with a monoclonal antibody to cytokeratin-8 (CK-8) was used. The immunoprecipitate was incubated for 24 h in the presence of different constituents involved in aggresome formation including ubiquitin, UBB+1, the proteasome inhibitor PS341, an ATP generating energy source, a deubiquitinating enzyme inhibitor, a purified proteasome fraction, and an E1–3 conjugating enzyme fraction. MB-like protein aggregates formed in the presence of ubiquitin, plus UBB+1 or PS341. These aggregates stained positively for CK-8. UBB+1, and a proteasome subunit Tbp7, as demonstrated on Western blots. A second approach was used to form MBs in vitro in cultured hepatocytes transfected with UBB+1 protein using Chariot. The cells were double stained using CK-8 and ubiquitin antibodies. The two proteins colocalized in MB-like aggregates. The results support the possibility that aggresome formation is a complex multifactor process, which is favored by inhibition of the proteasome and by the presence of UBB+1.

Short‐term dynamics of isotopic composition of leaf‐respired CO2 upon darkening: measurements and implications

Recent advances in understanding the metabolic origin and the temporal dynamics in delta(13)C of dark-respired CO(2) (delta(13)C(res)) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in delta(13)C(res) have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in delta(13)C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in delta(13)C(res) (ranging from 0 to 10 per thousand diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in delta(13)C(res) was negligible in all investigated species. Further, rapid post-illumination changes in delta(13)C(res) (30 min), which increased from 2.6 per thousand to 5 per thousand over the course of the day, were examined by positional (13)C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in delta(13)C(res). However, a Rayleigh fractionation of (12)C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in delta(13)C(res). These results are highly relevant since delta(13)C(res) is used in large-scale carbon cycle studies.

Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers.

Thrombin like activity of Asclepias curassavica L. latex: Action of cysteine proteases

Aim of the study To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. Materials and methods A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. Results The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen–agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Conclusion Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

Mining for new enzymes

Mining for new enzymes

Pleurotus species convert monoterpenes to furanoterpenoids through 1,4-endoperoxides

Enzymatic synthesis of furanoterpenoids from β-myrcene and related monoterpenes was observed using a solubilised enzyme fraction of mycelium lyophilisates of several Pleurotus species. The initial enzymatic step, the incorporation of molecular oxygen into the conjugated 1,3-diene moiety, was similar to a 2 + 4 cycloaddition of 1,3-dienes with dienophilic 1O 2, and was followed by a non-catalysed degradation sequence leading to the furans. The cyclic peroxides 3,6-dihydro-4-(2-(3,3-dimethyloxiran-2-yl)ethyl)-1,2-dioxine and 5-(3,6-dihydro-1,2-dioxin-4-yl)-2-methylpentan-2-ol were identified as key intermediates. Biotransformation of β-myrcene in 18O-labelled HEPES-buffer did not yield a detectable label in perillene, so a water addition to 3,10-epoxy-β-myrcenes as an alternative was ruled out. The pathway suggested presents a substantiated biogenetic scheme for the formation of monoterpenoid furans and opens biotechnological access to valuable natural flavour compounds, such as perillene and rosefurane. Only one metabolite, identified as the new natural compound 6-methyl-2-methylene-hept-5-enal, carried the 18O-label. The enzymatic formation of this compound through a 1,2-endoperoxide (3-(5-methyl-1-methylene-hex-4-enyl)-[1,2]-dioxetane) is suggested. The label may simply result from a chemical oxygen exchange between the carbonyl group and the 18O-labelled water.

Electrochemical and Biochemical Analysis of Ethanol Fermentation of Zymomonas mobilis KCCM11336

An electrochemical bioreactor (ECB) composed of a cathode compartment and an air anode was used in this study to characterize the ethanol fermentation of Zymomonas mobilis. The cathode and air anode were constructed of modified graphite felt with neutral red (NR) and a modified porous carbon plate with cellulose acetate and porous ceramic membrane, respectively. The air anode operates as a catalyst to generate protons and electrons from water. The growth and ethanol production of Z. mobilis were 50% higher in the ECB than were observed under anoxic nitrogen conditions. Ethanol production by growing cells and the crude enzyme of Z. mobilis were significantly lower under aerobic conditions than under other conditions. The growing cells and crude enzyme of Z. mobilis did not catalyze ethanol production from pyruvate and acetaldehyde. The membrane fraction of crude enzyme catalyzed ethanol production from glucose, but the soluble fraction did not. NADH was oxidized to NAD+ in association with H2O2 reduction, via the catalysis of crude enzyme. Our results suggested that NADH/NAD+ balance may be a critical factor for ethanol producton from glucose in the metabolism of Z. mobilis, and that the metabolic activity of both growing cells and crude enzyme for ethanol fermentation may be induced in the presence of glucose.

Kinetic parameters for thermal inactivation of soluble peroxidase from needles of Serbian spruce Picea omorika (Pančić) Purkynĕ

Thermal inactivation of peroxidase (POD) in an extract of Picea omorika (Pancić) Purkyne needles initiated by heat treatment was studied. This is the first study of this kind on a conifer species. Non-linear regression analysis was applied on the inactivation rate data, combining Mitscherlich and Arrhenius equations, treating time and temperature simultaneously as explaining variables. We determined the inactivation rate constant k, the Arrhenius energy of inactivation E and the remaining activity C(min) for the crude extract and for separated acidic and basic enzyme fractions, as well as for individual isoenzymes separated electrophoretically. A comparison of inactivation parameters for acidic and basic fractions shows that the thermal inactivation rate of the basic fraction is higher. The obtained value of inactivation energy for crude extract was between the values for acidic and basic isoenzyme fractions. One of the three analysed individual isoenzymes was characterised by a lower inactivation rate constant and higher inactivation energy. Another isoenzyme showed considerably higher level of remaining activity compared to the others, which identified it as the most resistant to high temperatures. The acquired values of Arrhenius energy of inactivation for POD in crude extract were intermediate, considering a range of POD values for various other plant species.

Characterization of Enzyme Endoxylanase Produced by Mutants Strains of Aspergillus Awamori K-1

ABSTRACTXylanolytic enzymes of microbial origin have received great attention due to their biotechnological utility and potential applications in a range of industrial processes but the observed effects vary depending on xylanase specificities.The goal of this investigation was to determine the endoxylanase enzyme production ability and molecular characteristics of fungal strains Aspergillus awamori K-1 and its mutant strains A59, A50, A45 and A60. The strains were cultivated in liquid culture with 1% wheat bran and 2% ground corn-cobs as inducer. Maximum enzyme production of parent strain Aspergillus awamori K-1 of 42,35 IU/ml and of mutant strains A59 - 96,91 IU/ml, A50 - 71,67 IU/ml, A45 - 74,04 IU/ml and A60 - 62,59 IU/ml was determined after 72–96 h of cultivation.Extracellular endoxylanase was partially purified from the culture filtrates of five strains, using molecular sieve chromatography with two type of Sephadex—G 50 and G100. The purification conditions were optimized and the main endoxylanase activity was determined in second fraction in case of Sephadex G100. The partial purified enzyme fractions showed the same band for all strains on SDS polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 32 kDa.

EFFECT OF TEMPERATURE ON THE ACTIVITY OF ACID PHOSPHATASES ISOLATED FROM CHLORELLA FUSCA, CLADOPHORA GLOMERATA AND PHORMIDIUM FAVEOLARUM AND THERMAL STABILITY OF THE ENZYMES

Temperature effect has been investigated in this study on the phosphatases isolated from three algae representing different groups; a unicellular freshwater green alga Chlorella fusca, a filamentous green macroalga Cladophora glomerata and a filamentous freshwater blue green microalga, Phormidium faveolorum. The temperature dependence of the enzyme activity could be summarized as that 60 oC was the optimum temperature for the enzyme activity in Cladophora glomerata as well as Chlorella fusca whereas Phormidium faveolorum had an optimum temperature of 70 oC for both isolated enzyme fractions, the cell wall-bound and the secreted extracellular. The thermal behavior of the phosphatases isolated from the tested algae showed toleration to the thermal activation when p-nitrophenyl phosphate (pNPP) was utilized as a substrate which has been investigated to be itself is not sensitive to high temperatures. The two fractions of the enzyme isolated from the algae exhibited high stability at high temperatures even above 80 oC

Isolation and characterisation ofBacillus amyloliquefaciens H2 glutamyl endopeptidase that is secreted in stationary phase of culture growth

The culture broth ofBacillus amyloliquefaciens H2 was used for isolation by chromatography on CM-cellulose and MonoS columns of proteinase that are secreted in early and late stationary phases of culture growth. Based on their inhibition by specific serine proteinase inhibitors and their action on specific chromogenic substrates and on oxidised B-chain of insulin, the enzymes were classified as glutamyl endopeptidases. The molecular weight of secreted enzyme is 26.5 kDa. We show that although the enzyme is secreted in different phases of culture growth, both enzyme fractions have similar enzymatic properties and amino acid content, but they were distinguished in.

Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.

Cysteine proteases from the Asclepiadaceae plants latex exhibited thrombin and plasmin like activities

In the present study we evaluated the presence of cysteine protease from the latex of four plants Asclepias curassavica L., Calotropis gigantea R.Br., Pergularia extensa R.Br. and Cynanchum puciflorum R.Br. belongs to the family Asclepiadaceae. Cysteine proteases from these plants latex exhibited both thrombin and plasmin like activities. Latex enzyme fraction in a concentration dependent manner induced the formation of clot in citrated blood plasma. Direct incubation of fibrinogen with latex enzyme fraction resulted in the formation of fibrin clot similar to thrombin enzyme. However prolonged incubation resulted in degradation of the formed fibrin clot suggesting plasmin like activity. Latex enzyme fraction preferentially hydrolyzed Aalpha and Bbeta chains of fibrinogen to form fibrin clot. Latex enzyme fraction also hydrolyzed the subunits of fully cross linked fibrin efficiently, the order of hydrolysis was alpha-polymer > alpha-chains > beta-chain and gamma-gamma dimer. Cysteine proteases from all the four Asclepiadaceae plants latex exhibited similar action on fibrinogen and fibrin. This study scientifically validate the use of plant latex in stop bleeding and wound healing by traditional healers all over the world.